Development of soluble TCR and soluble MHC/peptide compelx with high affinity for their ligands

• Project/Area Number: 08557026
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 展開研究
• Research Field: Immunology
• Research Institution: Kyushu University
• Principal Investigator: SASAZUKI Takehiko Medical Institute of Bioregulation, Kyushu University, Professor, 生体防御医学研究所, 教授 (50014121)
• Co-Investigator(Kenkyū-buntansha): FUKUI Yoshinori Medical Institute of Bioregulation, Kyushu University, Associate Professor, 生体防御医学研究所, 助教授 (60243961) ; KAMIKAWAJI Nobuhiro Medical Institute of Bioregulation, Kyushu University, Associate Professor, 生体防御医学研究所, 助教授 (90224659)
• Project Period (FY): 1996 – 1998
• Project Status: Completed (Fiscal Year 1998)
• Budget Amount: ¥12,000,000 (Direct Cost: ¥12,000,000); Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 1997: ¥2,500,000 (Direct Cost: ¥2,500,000); Fiscal Year 1996: ¥7,500,000 (Direct Cost: ¥7,500,000)
• Keywords: soluble TCR / soluble MHC / peptide complex / leucine zipper / off rate / affinity / ペプチド複合体 / MHC / GPIアンカー / PI-PLC / 高親和性 / Leucin Zipper

Research Abstract

We tried in this project to develop soluble MHC class II/peptide complex and soluble TCR with so high affinity as to directly identify and/or isolate their ligands. To facilitate the heterodimer formation, we connected leucine zipper sequence to the extracellular domain of alpha and beta chain of TCR or MHC class II.In addition, the antigenic peptide was covalently bound to MHC class II molecules. In each case, free cysteine was introduced at the C-terminal of one of the leucine zipper sequence, which was used for the target for the specific biotinylation.
Finally, we developed multivalent TCR or MHC class II/peptide complex by coupling the biotinylated molecule with streptavidin. Such multivatent TCR or MHC class II/peptide complex showed remarkably slow off-rate for their ligands in BIAcore analysis and detected the cells expressing the ligand by flow cytometry. Thus, this approach would have wide application for several immunological fields, including T cell development, autoimmunity, transplantation and tumor immunity.

Research Products

• [Publications] Nishimura H.: "MHC class II-dependent NK1.1+γβT cell are induced in mice by Salmonella infection." J.Immunol.in press. (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Gyotoku T.: "An endogenously processed self peptide and the coresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability." Eur.J.Immunol.28. 4050-4061 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukui Y.: "Highly restricted T cell repertoire shaped by a single major histocompatibility complex-peptide ligand in the presence of a single rearranged T cell recepter β chain." J.Exp.Med.188. 897-907 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Gapin L.: "Quantitative analysis of the T-cell repertoire by a single peptide/MHC complex." J.Exp.Med.187. 1871-1883 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukui Y.: "Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide liqand affected by its expression level in the thymus." Immunity. 6. 401-410 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishimura H,et al: "MHC class II-dependent NK1.1^+gammadeltaT cells are induced in mice by Salmonella infection." J.Immunol.(in press). (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Gyotoku T.et al.: "An endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability." Eur.J.Immunol.28. 4050-4061 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Fukui Y,et al.: "Highly restricted T cell repertoire shped by a single major histocompatibility complex-peptide ligand in the presence of a single rearranged T cell receptor beta chain." J.Exp.Med.188. 897-907 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Gapin L,et al.: "Quantitative analysis of the T-cell repertoire by a single peptide/MHC complex." J.Exp.Med.187. 1871-1883 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Fukui Y,et al.: "Positive and negative CD4^+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus." Immunity. 6. 401-410 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishimura H.: "MHC class II-dependent NK1,1+γδ T cells are induc ed in mice by Salmonella infection." J.Immunol.(in press). (1999)
• [Publications] Gyotoku T.: "An endogenously processed self peptide and the corresponding exogenous peputide bound to the same MHC class II molecule could be distinct ligands for TCR with di fferent kinetic stability." Eur.J.Immunol.28. 4050-4061 (1998)
• [Publications] Fukui Y.: "Highly restricted T cell repertoire shaped by a single major histocompatibility complex-peptide ligand inthe presence of a single rearranged T cell receptor β chain." J.Exp.Med.188. 897-907 (1998)
• [Publications] Gapin L.: "Quantitaive analysis of the T-cell repertoire by a single peptide/MHC complex." J.Exp.Med.187. 1871-1883 (1998)
• [Publications] Savoie,CJ.: "MHC class I bound peptides of a colon carcinoma cell line,a Ki-ras gene-targeted progeny cell line and a B cell line." Cancer Letters. (in press).
• [Publications] Tana T.: "A HLA binding motif-aided peptide epitope library:A novel library design for the screening of HLA-DR4-restricted antigenic peptides ecognized by CD4 T cells" J.Human Genet.(in press).
• [Publications] Ohmori M.: "Activated Ki-ras enhances sensitivity of ceramide-induced apoptosis without c-Jun NH2-terminal kinase/stress-activated protein kinase or extracellular signal-regulated kinase activation in human colon cancer cells." Cancer Research. vol.57. 4714-4717 (1997)
• [Publications] Kimura A.: "Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy." Nature Genet.vol.16. 379-382 (1997)
• [Publications] Furuse M.: "Suppression of serum-induced c-jun expression by activated Ki-ras in human colon cancer cells." Jpn.J.Human Genet.vol.42. 409-416 (1997)
• [Publications] Ohmori M.: "DNA typing of HLA class I (HLA-A) and class II genes (HLA-DR,-DO,and -DP) in Japanese patients with gastric cancer." Tissue Antigens. vol.50. 277-282 (1997)
• [Publications] Fukui Y.: "Differential requirement of MHC class II molecules expressed on hematopoietic cells for positive selection of CD4+ thymocytes in TCRab and TCRb transgenic mice." Int.Immunol.vol.9. 1385-1391 (1997)
• [Publications] Ishimoto T.: "In Vitro and In Vivo evidence for high frequency of I-Ab-reactive CD4+ T cells in HLA-DQ or HLA-DRA transgenic mice lacking endogenou MHC class I and/or class II expression." J.Immunol.vol.159. 3717-3722 (1997)
• [Publications] Fukui Y.: "Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus." Immunity. vol.6. 401-410 (1997)
• [Publications] Takeshita T.: "Identification of HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice." Jpn.J.Human Genet.vol.42. 225-232 (1997)
• [Publications] Fukui Y.: "Positive and negative CD4_+ thymocyte selection by a single MHC class II/peptide ligand in vivo affected by its expression level in the thymus." Immunity. (in press).
• [Publications] Takeshita T.: "Identification of HLA-DQ6-derived peptide recognized by mouse MHC class I H-2D^b-restricted CD8_+ T cells in HLA-DQ6 transgenic mice" Jpn.J.Human Genet.(in press).
• [Publications] Hori T.: "Japanese cedar pollinosis and HLA-DP5." Tissue Antigens. 47. 485-491 (1996)
• [Publications] Min W.: "Identification of an epitope for T cells correlated with antibody response to hepatitis B surface antigen in vaccinated humans." Human Immunol.46. 93-99 (1996)
• [Publications] Mineta M.: "Contribution of HLA class I and class II alleles to the regulation of antibody production to hepatitis B surface antigen in humans." Int.Immunol.8. 525-531 (1996)
• [Publications] Date Y.: "DNA typing of the HLA-A gene:population studay and identification of four new alleles in Japanese." Tissue Antigens. 47. 93-101 (1996)
• [Publications] Tamai H.: "Association between the DRB1^<**>08032 histocompatibility antigen and methimazole-induced agranulocytosis in Japanese patients with Graves Disease." Annals of Internal Medicine. 124. 490-494 (1996)
• [Publications] Yasunaga S.: "Different contribution of HLA-DR and-DQ genes in susceptibility and resistance to insulin-dependent diabetes mellitus(IDDM)." Tissue Antigens. 47. 37-48 (1996)